Most workers in the field consider loss of synapses to be the earliest correlate of cognitive loss in AD. Meanwhile, although APOE4 is the largest risk factor for Alzheimer's disease (AD), the mechanisms by which it increases risk are not understood. Similarly, the mechanisms for protection by APOE2 remain unknown. The present project is aimed at understanding APOE mechanisms in a key neuronal compartment, synaptic terminals. Synaptic terminals are not well-resolved at the level of light microscopy, which makes accurate determination of synaptic pathways technically difficult; therefore, my laboratory has developed methods for flow cytometry analysis of synaptosomes prepared from cryopreserved human and rodent tissue. This technique is used together with more routine biochemical assays and allows 1) study of populations of individual terminals (5-10,000 particles/sample) and 2) absolute quantification of synaptic A?, phosphorylated tau (p-tau), and lipid and mitochondrial markers in postmortem human cases and in transgenic disease models. The long term goal of this project remains to determine APOE genotype effects on synaptic pathways leading to A? accumulation and clearance. Our working hypothesis is that synaptic A? in surviving, 'sick' synaptic terminals represents a pre-deposit accumulation that contributes to synaptic dysfunction, and that APOE modulates clearance vs. early deposition pathways within synapses. Synaptosomes from three model systems will be used for Aims 1 and 2: i) postmortem staged AD cases and aged control cases, ii) young targeted replacement (TR) mice expressing human apoE2,3, and 4, and iii) aged TR mice. Young TR mice have low levels and aged TR mice have moderate levels of diffuse A?, avoiding confounds from the high A? level in some mouse models. Aim 1 will determine the effect of APOE on A? level, apoE level and lipidation, and on synaptic lipids in cortical synapses. Aim 2 will determine the effect of APOE and A? level on mitochondrial function, downstream tau pathology and autophagic flux. Aim 3 is in vitro, and will examine the contribution of APOE genotype and apoE level and lipidation to mitochondrial function and A? levels in cortical synaptosomes from aged TR mice. Synaptosomes will be treated with highly lipidated vs physiologically lipidated apoE, with and without A?42. Because apoE is a promising therapeutic target, the proposed experiments to clarify the role of APOE in the synaptic compartment are urgently needed in order to understand and predict the effects of apoE-directed therapeutics.